Telomeres are higher order nucleoprotein structures that physically cap the chromosome terminus and help to preserve genome integrity. In cells with unlimited proliferative capacity, including 95% of human cancers, telomeres are maintained by telomerase. By contrast, telomerase is absent from most of the normal soma and telomeres shorten until chromosome ends become uncapped and indistinguishable from double strand breaks. End-to-end fusions are induced, ultimately leading to cell cycle arrest. Here we propose to exploit Arabidopsis to investigate essential components of the telomere cap in a genetically tractable higher eukaryote. Arabidopsis has an exceptionally high tolerance for telomere dysfunction. This finding, coupled with its facile genetics, completely sequenced genome, and arsenal of transgenic tools offer unique opportunities for investigating essential genes in telomere biology. In this renewal application, we will focus on the recently discovered Protection of telomeres 1 (Pot1) protein, which binds the extreme terminus of the chromosome and plays a central role in chromosome end protection and telomere length regulation. Arabidopsis encodes three strikingly different Pot proteins, AtPot1, AtPot2 and AtPot3, which appear to be functionally non-redundant. We hypothesize that the separation of function of Arabidopsis Pot proteins derives from their distinct interactions with telomeric DNA and with other protein components of the telomere. The proposal is comprised of five Specific Aims. The first two focus on defining the interactions of Pot proteins with telomeric DNA in vitro and in vivo, and elucidating interaction partners in the telomere complex in vivo. For the last three Aims, we will exploit a combination of genetic and biochemical approaches to examine the mechanism of positive regulation of telomerase by AtPot1 (Aim 3), the contribution of AtPot2 to chromosome end protection (Aim 4), and the apparent developmental regulation of AtPot3 as it impacts this protein's function at telomeres (Aim 5). As part of Aim 5, we will also test the hypothesis that telomeres are uncapped in a specific stage of the plant life cycle. Given the strong conservation in telomere architecture and composition in plants and humans, these studies should uncover mechanisms common to all higher eukaryotes.